The precise role of the majority SCF-based E3 ligases, including F box proteins, has not been established. The observation that FBXL2 is repressed in human lung adenocarcinoma (Malard et al 2007
) raises the possibility that it might regulate molecular programs involved in neoplasia, such as cell cycle progression. Here, we show that both ectopically expressed and endogenous FBXL2 regulate viablity and proliferation of transformed or tumorogenic epithelia by ubiquitin-mediated degradion of cyclin D3. We also observe opposing activities of SCFFBXL2
and a ubiquitous calcium sensor, CaM, on cyclin D3 polyubiquitination and degradation. FBXL2 functions as the receptor component of a prototypical SCF ubiquitin E3 ligase that targets a CaM binding signature; this represents a unique property of FBXL2 as other F-Box proteins recognize phosphoserine or phosphothreonine sites within target substrates (Hansen et al 2004
, Liu et al 1999
, Watanabe et al 2005
). For example, the well-studied family member cyclin D1 harbors a key site (Thr286
) for recognition by the E3 ligase subunits FBXO4, FBXW8, and FBXO31 (Lin et al 2006
, Okabe et al 2006
, Santra et al 2009
). Although this Thr site is conserved among the D cyclins and might also be used as a targeting signal by cyclin D3 for other F box proteins, FBXL2 did not utilize this signature for its substrate. This was evidenced by the ability of both dephosphorylated cyclin D3 and a related point mutant (cyclin D3T283A
) to bind and be ubiquitinated by FBXL2 (Figs. S10–S12
). Rather, FBXL2 docks within a consenus IQ signature within its substrate to facilitate ubiquitination. This was evidenced by ectopic expression of point mutants of cyclin D3 within the IQ motif that were resistant to ubiquitination and were sufficient to extend cyclin D3 half-life (). Whether FBXL2 uses this mode of targeting to other substrates requires additional investigation, but these results provide the first evidence that this F-box protein appears to be a major regulator of cyclin D3 lifespan and thus might serve as a key growth inhibitory signal.
F-box protein mediated ubiquitination and proteasomal degradation of cyclin D3 resulting in G2/M arrest was unexpected, as these cyclins predominantly regulate G1/S transition and knockdown of cyclin D3 has been shown to induce G1 arrest (Sicinska et al 2003
). However, cyclin D3 may have a dual role in also mediating G2/M phase progression (Fang et al 2002
, Zhang et al 2002
). Thus, in lymphoblastic cells that selectively and highly expressed cyclin D3, its knockdown was expected to result in G1 arrest given the role of D-type cyclins in G1-S progression. However, in our studies, MLE cells express all three D-type cyclins, and overexpression of FBXL2 selectively down-regulated cyclin D3, but not cyclin D1; hence because of cyclin D redundancy G1/S phase progression is preserved and G2/M arrest was observed. In addition to this redundancy, the G1/S phase may also be less prone to FBXL2-induced blockade because of lower FBXL2 levels during interphase and higher levels of CaM and cyclin D3.
As above, cyclin D3 is also a multi-functional protein that directly interacts with and confers activity for CDK11p58 (Duan et al 2010
, Zhang et al 2002
), a key cyclin-dependent kinase that controls centrosome maturation and bipolar spindle formation (Petretti et al 2006
). As with ectopic FBXL2 in cells, knockdown of CDK11p58 results in G2 arrest and apoptosis; significant CDK11 depletion results in misaligned and lagging chromosomes, permanent mitotic arrest, and cell death (Hu et al 2007
). Hence, SCFFBXL2
directed ubiquitination and degradation of cyclin D3 would potentially impair its association with CDK11p58 and reduce its activity. One additional function of CDK11 is to recruit Polo-like kinase 4 (PLK4) and Aurora A to the centrosome that also regulate mitotic events and chromosomal stability (Petretti et al 2006
). Knockout of PLK4 or expression of a defective mutant Aurora protein also results in apoptosis (Rosario et al 2010
) (Glover et al 1995
). Collectively, these results suggest that one explanation for G2/M phase delay and apoptosis might involve SCFFBXL2
inactivation of CDK11p58 by ubiquitination and depletion of cyclin D3. This mechanism would dislocate PLK4 and Aurora A causing cell cycle arrest (). In support of this, ectopic expression of FBXL2 does not decrease CDK11 protein levels but reduces assembly of the cyclin D3: CDK11p58 complex and binding of PLK4 and Aurora A (data not shown). There also exist some differing effects on cycle progression after silencing cyclin D2 or cyclin D3. Knockdown of cyclin D3, but not cyclin D2, resulted in G2/M arrest. Because both cyclins are highly conserved and exert some redundant functions, a more modest phenotype observed with cyclin D2 knockdown might be because of compensation by cyclin D3. This would occur especially if lower levels of cyclin D2 are present in MLE cells compared to cyclin D3 as seen with A549 cells and CHO cells (Fig. S9
FBXL2 induction of cell cycle arrest is opposed by CaM
We have uncovered functionally distinct domains that govern molecular interplay between the effectors, FBXL2 and CaM, and their putative targets, cyclin D3. First, cyclin D3 harbors a canonical IQ motif typical of calcium independent CaM binding proteins (). Accordingly, both FBXL2 and CaM target this signature within cyclin D3 and bind in the absence of calcium (, ). Structural analysis predicts a α-helix for the IQ signature with cyclin D3 (LQLLGTVCLL). Interestingly, cyclin D1 contains a modified sequence (LQLLGATCMF) with substitution of a Thr for a hydrophobic residue at position seven. This could explain our findings that ectopic expression or knockdown of FBXL2 does not affect cyclin D1 levels (Fig. S8
). Alternatively, this could be because cyclin D3 is the dominant D-type cyclin within the MLE cells, thus prone to FBXL2 targeting.
Our data indicate that calcium availability differentially regulates the interactions between the F box protein, CaM, and the FBXL2 substrate. In our prior studies, the presence of calcium did not alter the molecular interaction between CaM and cytidylyltransferase (Chen and Mallampalli 2007
) and yet CaM binding with cyclin D3 was completely interrupted by even 1 μM calcium. In both cases calcium promotes FBXL2 interaction with its substrates, although FBXL2 still associates with cyclin D3 in the absence of calcium. Physiologically, these molecular interactions might depend upon subcellular compartmentalization of binding partners and availability of calcium signals within these compartments. For example, cytidylyltransferase is an amphitrophic enzyme that largely exists in the cytoplasm in lung cells whereas cyclins are nuclear (Ridsdale et al 2001
). Hence, cytidylyltransferase would be predicted to be protected by CaM in settings when calcium levels are very low, but during sepsis when cytosolic calcium currents fluctuate, it might be prone to ubiquitination by calcium-activated SCFFBXL2
(Chen et al 2011
). In the nucleus, the physiological levels of calcium will also likely regulate these molecular interactions during cell division. Although cyclin D3 binds CaM in the absence of calcium, a modest increase in calcium (~1000nM) almost totally disrupts cyclin D interaction with CaM (, ) and calcium increases cyclin D3 binding to FBXL2 (). However, during the G1/S phase, CaM binds cyclin D3 () when low levels of calcium (20–40 nM) are typically present in the nucleus providing an environment conducive for these interactions (Korkotian and Segal 1996
). Moreover, during prophase the nuclear envelope is disrupted, chromosomal condensation occurs, and nuclear contents are transiently exposed to higher calcium concentrations within the cytosol (200–1000 nM) (Brown and Shoback 1984
, Pszczolkowski et al 1999
). The prediction is that these higher calcium concentrations would trigger CaM dissociation from cyclin D3 to enhance cyclin vulnerablity for SCFFBXL2
mediated ubiquitination. Here, calcium might play a more important role in destabilizing cyclin D3 by releasing CaM and recruiting SCFFBXL2
to the IQ motif.
Our data suggest that cyclin D3 availability required for cell cycle progression will also depend on the relative binding affinities between FBXL2, CaM, and their targets. Each of these proteins was demonstrated to interact in vitro (). Of note, despite ectopic expression of an Adv5-CaM in cells, this was inefficient at restoring cyclin D3 levels when co-expressing an FBXL2 mutant that lacks ability to interact with CaM. These results suggest that CaM's ability to act as a decoy to directly antagonize and sequester the F-box protein represents a mechanistically relevant interaction in addition to FBXL2 and CaM intermolecular competition for occupancy within the cyclin IQ motif. However, our isothermal calorimetry studies demonstrating very low binding constants between CaM and cyclin D3 (Kd= 0.31 μM, ()) versus relatively higher values between CaM and FBXL2 (Kd= 0.81 μM, data not shown) suggest that CaM competition with FBXL2 might be a more functionally relevant mechanism in vivo.
The data presented here suggest that tight interplay between F-box protein FBXL2 and CaM will regulate mitotic events through control of cyclin D3 abundance. CaM plays a vital role in centrosome formation during mitosis by interacting with the centrosome protein, CP110; mutant CP110 that lacks ability to bind CaM leads to failure of cytokinesis (Tsang et al 2006
). Counter-intuitively, CaM fails to protect cyclin D3 during mitosis thereby potentially contributing to SCFFBXL2
-induced ubiquitination and degradation. As stated above, cyclin D3 dependent CDK11p58 activity is also essential for mitosis, but excessive CDK11p58 levels repress cellular proliferation and induce apoptosis (Duan et al 2010
). Hence, the dissociation of CaM from cyclin D3 and its targeting by the SCFFBXL2
complex during the transition to mitosis might be an exquisite mechanism to balance CDK11p58 levels thereby regulating cell proliferation.